During the embryonic period of CNS development NSCs are considered to be the proliferating cellular compartment in the neuroepithelium that both expands itself through self-renewal via symmetrical divisions and generates lineage-restricted progenitors via asymmetrical divisions. Progenitor progeny also divide both symmetrically and asymmetrically and ultimately differentiate into the neural phenotypes composing different stages of CNS development. Although the seminal biology of NSCs has come under intensive investigation, there is still no consensus regarding exactly which cells are actually NSCs, since specific markers do not exist. So, neuroepithelial cells are widely used as the primary source of NSCs, and often as if they all were NSCs. Thus, there is a general consensus that further elucidation of NSC biology first requires their identification since this could provide direct experimental access to them for prospective rather than retrospective investigation. [unreadable] During FY2008 we focused entirely on relating our cell phenotyping strategy to all of the available markers currently used in the field of neural stem/progenitor cell research. Different investigators use different markers and methods to try to identify putative NSCs from retrospective analyses of heterogeneous cell populations exhibiting generic functional endpoints (self-renewal without differentiation and differentiation along neuronal, astroglial and oligoglial lineages). The results have led to a continuing lack of consensus regarding the seminal properties of NSCs. So, many types of putative NSCs are lumped together because specific markers for NSCs remain elusive and investigators have realized that lineage-restricted progenitor phenotypes exhibit seminal properties commonly attributed to NSCs. Our specific aim has been to resolve NSC and progenitor populations using all-inclusive immunostaining protocols, which incorporate multiple NSC-associated markers that are commercially available and accessible to those in the field.[unreadable] Previously, we had identified NSCs at the onset of cortical development based on their lack of surface expression of known neural markers, including tetanus toxin (TxTx) fragment C and cholera toxin B complex ganglioside binding sites, A2B5 (GD3 ganglioside) and CD15/LeX/SSEA-1 (a trisaccharide). We have now included a much more comprehensive assortment of surface (CD133, NG2, PSA-NCAM, CD24, CD57, NGFR p75, integrin alpha 3, integrin alpha 6, integrin beta 1), cytoskeletal/cytoplasmic (beta 4 tubulin, alpha 1 tubulin, Aldefluor) and nuclear markers (HuD, Ngn1, Ngn3, Mcm2, FoxG1, Dlx2, Olig2, Pax6, Prox1, Sox1, Sox2, Hoechst), which are used in the NSC/progenitor field, in order to comprehensively phenotype cortical NSCs that we had earlier isolated by negative selection. We combined pairs of alpha and beta integrin subunits with CD57, a pan-specific marker of CD15+ CD24+ CDw60+ PSA-NCAM+ A2B5+ neural progenitors, and TnTx, a pan-specific marker of all neuronal progenitors and post-mitotic neurons. This novel combination of markers provided us with an unprecedented level of resolution revealing the lineal relationships among all the major populations throughout cortical development. NSCs were identified as CD57- TnTx- cells expressing or co-expressing alpha and beta integrin subunits. These cells decreased in relative abundance while increasing in absolute abundance during the embryonic period as they either self-renewed, died via apoptosis or differentiated into different CD57+/- and TnTx+ progenitor phenotypes. We also included a live-cell DNA stain and used flow cytometry to quantify total DNA content. The most proliferative subsets were found in CD57+ cells co-expressing alpha and beta integrin subunits, while the most abundant post-mitotic cells were found in the CD57- TnTx+ population devoid of either integrin subunit. This comprehensive surface phenotyping strategy provides, for the first time, a complete ex vivo account of the proliferating, quiescent/differentiating and apoptotic phases that accompany the different intermediate stages of the various lineage progressions derived from CD57- TnTx- NSCs.[unreadable] [unreadable] Analysis of the four bivariate FACS plots (CD57+/- TnTx+/-) associated with the four integrin subunit-labeled populations (alpha+/- beta+/-) revealed clear linkages among each of the neural populations either devoid of markers (CD57- TnTx- NSCs) or expressing either one or both markers. We conclude that NSCs give rise to both neuronal progenitors (CD57- TnTx+) and neural progenitors (CD57+ TnTx-), which, in turn, produce other neuronal progenitors (CD57+ TnTx+). [unreadable] [unreadable] The surface expressions/coexpressions of these markers allowed us to sort select populations for analysis of their seminal properties using clonal cultures. The results obtained in vitro closely recapitulate the lineage progressions inferred from FACS analyses of populations phenotyped ex vivo, indicating that seminal properties are effectively preserved and so experimentally accessible for cellular and molecular studies. It is clear that the seminal properties, including self-renewal, apoptosis, and differentiation, are all expressed by lineage-negative NSCs, CD57+ TnTx- neural progenitors and CD57- radial glia. Thus, each of these phenotypes exhibits similar, but not identical seminal properties typically ascribed to NSCs. We also quantified the distributions of three other surface markers (CD133, NG2, NGFR p75) in the context of the beta integrin subunit, CD57 and TnTx. Each of these markers is expressed by rare populations throughout development. CD133+ cells primarily reside at the ventricular interface in proliferating cells and may only be expressed at specific stage(s) of the cell cycle. The other two markers are mostly expressed by cells devoid of neural markers. Survey of several cytoplasmic markers revealed that alpha 1 tubulin is primarily expressed by CD57+ TnTx- neural progenitors, while beta 4 tubulin is restricted to oligodendroglial progeny. Neither Aldefluor labeling nor Hoechst dye exclusion were specific, since subpopulations of CD57- TnTx- NSCs, CD57- TnTx+ neuronal progenitors and CD57+ TnTx- neural progenitors were found to be Aldefluor+ and excluded Hoechst dye. Survey of NSC-associated nuclear markers (transcription factors, TFs) showed that Pax6, Sox1 and Sox2 label the majority of CD57- TnTx- NSCs and CD57+ TnTx- neural progenitors throughout cortical development. These TFs are also expressed by CD57- radial glia, but are down-regulated among the differentiating neuronal phenotypes generated by these populations. Other TFs (HuD, Ngn1, Ngn3, Prox1) emerge primarily among differentiating neuronal progenitors, especially those derived from CD57+ TnTx- progenitors, while Mcm2 and FoxG1 are co-expressed by these cells. [unreadable] In sum, lineage-negative NSCs predominate at the earliest stages of corticogenesis and generate CD57- TnTx+ and CD57+ TnTx- progenitors, which differentiate into transiently expressed pioneer neurons and transiently expressed Cajal-Retzius neurons, respectively. Later, CD57+ TnTx- progenitors generate the permanent pyramidal neurons composing the cortical layers. NSCs self-renew and differentiate into radial glia and astroglia. All of these cells coexpress integrin subunits at varying levels. Our analysis has revealed 20 distinct populations with some transiently appearing, then disappearing and others persisting during cortical development. Using multi-marker phenotyping thus provides a complete characterization of the emerging populations and reveals the dynamic lineal relationships that exist among them. This novel, all-inclusive framework is based on results that have been derived from using a tool kit of commercially available reagents, thus making the strategy accessible to all interested investigators.